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BACKGROUND: Traditionally, blood group typing has been performed with serologic techniques, the classical method being the hemagglutination test. Serotyping, however, may present important limitations such as scarce availability of rare antisera, typing of recently transfused patients, and those with a positive direct antiglobulin test. Consequently, serologic tests are being complemented with molecular methods. The aim of this study was to develop a low-cost, high-throughput method for large-scale genotyping of red blood cells (RBCs). STUDY DESIGN AND METHODS: Single-nucleotide polymorphisms associated with some clinically important blood group antigens, as well as with certain rare blood antigens, were evaluated: Jka/Jkb, Fya/Fyb, S/s, K/k, Kpa/Kpb, Jsa/Jsb, Coa/Cob, and Lua/Lub. Polymerase chain reaction (PCR)-amplified targets were detected by direct hybridization to microspheres coupled to allele-specific oligonucleotides. Cutoff values for each genotype were established with phenotyped and/or genotyped samples. RESULTS: The method was validated with a blind panel of 92 blood donor samples. The results were fully concordant with those provided by hemagglutination assays and/or sequence-specific primer (SSP)–PCR. The method was subsequently evaluated with approximately 800 blood donor and patient samples. CONCLUSION: This study presents a flexible, quick, and economical method for complete genotyping of large donor cohorts for RBC alleles. [ABSTRACT FROM AUTHOR]